1. Field of the Invention
The present invention relates generally to the transfection of eukaryotic cells, the regulation of gene expression, and gene therapy. In particular, the present invention relates to novel compositions and uses thereof including, but not limited to: the regulation of nucleic acid expression, the transfection of eukaryotic cells, gene therapy, the creation of transgenic animals, the biological production of pharmaceuticals, and the treatment of a variety of human diseases and disorders.
2. Description of Related Art
The State of the Technology: Gene Transfer
One of the most utilized and important techniques employed in the biological sciences today is the transfer of foreign nucleic acids into cells in vitro and in vivo. This technology is the foundation for gene therapy. A primary obstacle to the successful implementation of gene transfer technology is that cellular membranes provide a significant barrier to the translocation of nucleic acid. Currently, techniques exist for the transfer of nucleic acids across the cytoplasmic membrane of prokaryotic (transformation) and eukaryotic cells (transfection) including chemical, physical, and biological methods such as: calcium phosphate co-precipitation, DEAE dextran treatment, electroporation, microinjection, biolistic bombardment, viral infection, and liposomal based methods. The nuclear membrane of eukaryotic cells, however, proves to be a more formidable challenge. This membrane is a barrier to the passive movement of macromolecules larger than 15,000 kDa. Empirically, transfection protocols are limited to rapidly dividing cells. The hypothesis for these observations is that transfected nucleic acids have access to the nuclear compartment only when the nuclear membrane is dissolved during mitosis. Without a nuclear membrane, the transfected nucleic acids are thought to distribute throughout the volume of the cell, and a portion of these nucleic acids might remain in the nucleus after the nuclear membrane reforms.
Various strategies have been attempted to circumvent an intact nuclear membrane during transfection. Several viral vectors are under study as gene transfer agents, but all of them have major disadvantages. Viral vectors are a biohazard, it is usually necessary to employ procedures to limit viral replication by eliminating certain viral genes and using helper virus strains, the gene of interest must be cloned into the viral vector, and adeno-associated vectors (AAV) are difficult to produce in large quantities and have a limited capacity for accepting large transgenes. The transfection efficiency of most viral vectors is dependent upon proliferation of the host cell which, again, limits the utility of such vectors. Replication deficient adenoviral vectors have been used extensively in the lungs, but trigger an acute inflammatory response and a chronic immune response. Retroviral vectors, which insert randomly into the host genome, have the potential to disrupt normal genes and carry a risk of inducing malignancies. Thus, viral vectors show limited utility for in vivo therapy.
Several non-viral strategies to circumvent an intact nuclear membrane during transfection have been investigated, but none has proven to be efficient. One attempt is described in U.S. Pat. No. 5,827,705 to Dean. The Dean U.S. Pat. No. 5,827,705 patent utilizes a nucleic acid segment, referred to as a nuclear transport sequence (NTS), found to promote the transport of plasmid DNA into the nucleus of several cell types following microinjection of the plasmid into the cell. This approach has several major drawbacks including the need to clone the gene of interest into this specialized plasmid and the need for microinjection into individual cells as part of the transfection procedure.
Another attempt was described by Rossi et al. (1993). A plasmid DNA was covalently linked to a nuclear localization sequence (NLS) for nuclear import; however, this system damages the DNA making it non-viable for expression or replication.
Still another transfection method was described in U.S. Pat. No. 5,807,746 to Lin et al. In the Lin U.S. Pat. No. 5,807,746 patent, an importation competent signal peptide (ICSP) was demonstrated to promote the passage of a biologically active peptide through the cytoplasmic membrane. The ICSP was also made in conjunction with an NLS (ICSP-NLS). The ICSP-NLS promoted the passage of the biologically active peptide into the nuclear compartment. The Lin U.S. Pat. No. 5,807,746 patent suggested methods for linking nucleic acids to the ICSP-NLS using charge association or covalent linkage by thioester bonds. However, charge-association is too weak of an interaction for efficient transfection (Fritz et al. (1996) Human Gene Therapy 7:1395-1404) and covalent linkage eliminates the functionality of the nucleic acid (Rossi et al. (1993) Molecular and General Genetics 239:345-353).
A second major challenge to the successful implementation of gene transfer technologies involves the phenomenon of gene silencing or transient expression of transferred nucleic acids. This effect is characterized by the termination of transgene expression within three or four days of transfection. One explanation for this phenomenon is that the foreign nucleic acids are transported out of the nucleus by a cellular process (Alwine (1985) Mol Cell Biol 5:1034-1042). The common redress in vitro for transient expression is to employ selection methods to kill cells that do not express a co-transfected detoxifying agent. What is needed is a system that retains viable nucleic acids and plasmids in the nucleus which are capable of long-term expression without the need for toxic selective agents.
The State of the Technology: Peptide Nucleic Acid
Peptide nucleic acids (PNAs) are analogs of nucleic acids in which the ribose-phosphate backbone has been replaced with a backbone that is held together by amide bonds as described in U.S. Pat. No. 5,539,082 to Nielsen et al. PNAs have several interesting features, including the ability to hybridize to complementary DNA, RNA, or peptide nucleic acid (PNA) sequences. The hybridization of a PNA to a complementary sequence is generally greater than that of nucleic acid to nucleic acid hybrids (Good and Nielsen (1997) Antisense and Nuc Acid Drug Dev (7)431-7). Furthermore, PNAs are resistant to proteases and nucleases, and exhibit an ability to modulate transcription in a positive direction when hybridized to the non-coding strand of a promoter region and in a negative direction when hybridized to the coding strand of a promoter region (N. E. Mollegaard (1994) Proc. Natl. Acad. Sci. 91(9)3892-3895; Good and Nielsen, 1997). Specific PNA molecules have been used as antisense oligonucleotides (Good and Nielsen (1998) Nature Biotechnology (16)355-358), transcription enhancers and transcription repressors (N. E. Mollegaard, 1994; Good and Nielsen, 1997; Wang et al. (1999) Nuc Acids Res 27:2806-2813), and PCR(trademark) clamping reagents (U.S. Pat. No. 5,656,461 to Demers).
Deficiencies Inherent in the State of the Technology
Current transfection methods are inefficient. Particularly lacking are methods for transfecting cells with an intact nuclear membrane. Methods for transfecting mitotic cells (in which the nuclear membrane has dissolved) are also limited because only a few percent of cells in a given population are mitotic and because transgene silencing eliminates expression within a few days without selective agents and the additional transfection of selection resistance genes. Improved transfection methods would benefit both basic scientific research and clinical medicine. The limited success of gene therapy thus far can be attributed to procedures that target actively dividing cell populations and likely achieve expression in less than one percent of targeted cells. Improved transfection methods that permit the efficient transfection of nucleated cells would immediately yield dramatic improvements in basic science, clinical medicine, and especially gene therapy. Benefits would include: the ability to express a gene in a greater percentage of any population of cells (including actively dividing cell populations), the ability to transfect all cell types (including quiescent, non-dividing, differentiated, and the like), reducing the dosage of gene therapy required to achieve an effect, reducing toxicity, and enhancing opportunities for routes of administration. In addition, it would be helpful if such a system were capable of regulating expression of any endogenous or exogenous nucleic acid.
The present invention provides compositions and methods for use thereof that overcome deficiencies in the prior art related to, but not limited to: transfection of eukaryotic cells, regulation of gene expression, regulation of epigenetic gene expression, production of nucleic acid and protein pharmaceuticals, and gene therapy.
The present invention is directed to a composition comprising a nuclear localization sequence (NLS) and a peptide nucleic acid (PNA) (the combination referred to herein as an NLS-PNA) which is useful for transferring a nucleic acid into the nucleus of a cell; for regulating an expression of the transferred nucleic acid, of an endogenous nucleic acid, or an epigenic nucleic acid; for the creation of transgenic animals; and as a gene therapy agent. The NLS-PNA is also useful in conjunction with cells that were previously refractory to transfection.
In certain embodiments, the PNA is annealed to a complementary region of the nucleic acid (NA) to form a stable NLS-PNA-NA. The NLS-PNA-NA can be transfected into the cytoplasm of a nucleated cell using standard methods. The NLS transports the entire NLS-PNA-NA from the cytoplasm into the nuclear compartment and maintains the localization of the NLS-PNA-NA in the nuclear compartment. Annealing of the PNA to the nucleic acid does not damage the nucleic acid; thus, the nucleic acid is capable of expressing a gene product. The nucleic acid may include a coding strand and a non-coding strand. Annealing of the PNA to the non-coding strand, especially in the region of the promoter, stimulates expression of the gene product. In certain embodiments, an NLS-PNA can be used to transfer a nucleic acid into the nucleus of an embryonic stem cell for the creation of a transgenic animal.
An NLS-PNA can be used in any gene therapy application as a rescue agent to enhance or suppress expression of the gene therapeutic by targeting the PNA to a non-coding or a coding strand of the gene therapeutic, respectively, especially in the region of a promoter.
An NLS-PNA can also be used to regulate an expression of a nucleic acid segment, including a gene, in a nucleus of a cell. The NLS-PNA can be transfected into the cytoplasm of a nucleated cell using standard methods. The NLS transports the entire NLS-PNA from the cytoplasm into the nuclear compartment and maintains the localization of the NLS-PNA in the nuclear compartment. The PNA portion of the NLS-PNA is designed to be complementary to a region on the targeted nucleic acid segment. The PNA anneals to the complementary region and, accordingly, enhances or suppresses an expression of the nucleic acid segment. Multiple nucleic acid segments (including introns and exons of a gene or multiple genes) can be targeted at once with one NLS-PNA or with distinct NLS-PNAs. In certain embodiments, annealing of the PNA to a viral nucleic acid inhibits an activity of the viral nucleic acid or an expression of a viral gene product. It is contemplated that the nucleic acid includes, but is not limited to, single-stranded nucleic acid, double-stranded nucleic acid, DNA, RNA, and single- or double-stranded viral nucleic acid.
In certain embodiments, the NLS-PNA is introduced into the cytoplasm of the cell by combining the NLS-PNA with a membrane transport sequence (MTS). This novel composition is referred to herein as an MTS-NLS-PNA, but the elements can be combined in any order. An MTS and its use in translocating a cytoplasmic membrane are described in U.S. Pat. No. 5,807,746 to Lin et al., incorporated herein by reference.
It is an object of the present invention to provide compositions useful as transfecting reagents.
It is a further object of the present invention to provide a system designed to transfer a nucleic acid into the nucleus of a eukaryotic cell including, but not limited to: a yeast cell, an insect cell, a plant cell, an animal cell, a mammalian cell, a mouse embryonic cell, a human cell, and a hybrid cell.
It is an additional object of the present invention to provide a system designed to transfect a nucleic acid into a nucleus of a eukaryotic cell including, but not limited to: a cell with an intact nuclear membrane, a non-dividing cell, a quiescent cell, a terminally differentiated primary cell, an embryonic stem cell, and the like.
It is another object of the present invention to enhance the transfection efficiency for transfection of dividing populations of eukaryotic cells.
It is still another object of the present invention to provide a system designed to stimulate the expression of a nucleic acid transfected into a eukaryotic cell, especially without the need to utilize toxic selection agents or resistance genes.
It is yet another object of the present invention to provide a system designed to regulate an expression of a nucleic acid transfected into a nucleus of a eukaryotic cell, including the rescue (treatment) of an overabundant or an insufficient gene therapy.
It is yet a further object of the present invention to provide a system designed to regulate an expression of a nucleic acid segment, wherein the nucleic acid segment is in the nucleus of a eukaryotic cell, and wherein the regulation includes stimulation of expression or inhibition of expression. One aspect of the present object includes the prevention and treatment of a medical condition or disease through the medically relevant regulation of the nucleic acid segment. Another aspect of the present object includes kits designed for use in the prevention or treatment of a medical condition or disease through the medically relevant regulation of the nucleic acid segment.
It is also an object of the present invention to provide compositions and methods useful for in vitro transfection, in vivo transfection, ex vivo transfection, in situ labeling, diagnostic tests, genetic therapy, gene therapy, treatment of medical conditions, and the creation of transgenic animals. One aspect of the present object comprises kits designed for in vitro transfection, in vivo transfection, ex vivo transfection, in situ labeling, diagnostic tests, genetic therapy, gene therapy, treatment of medical conditions, and the creation of transgenic animals.
Accordingly, the present invention includes a composition comprising a nuclear localization sequence (NLS) and a peptide nucleic acid (PNA). The preferred method of making an NLS-PNA comprises solid phase synthesis of a single sequence of amino acids residues (for the NLS portion) and peptide nucleic acid residues (for the PNA portion), wherein the residues are linked by peptide bonds. A PNA comprises a nucleic acid analog in which nucleic acid bases are attached to a peptide backbone through a suitable linker, as described in U.S. Pat. No. 5,539,082 to Nielsen et al., incorporated herein by reference.
Accordingly, the present invention provides methods for transferring a nucleic acid into a nucleus of a cell using compositions disclosed herein. In certain embodiments, a PNA is annealed to a complementary region of a desired nucleic acid and transfected into the cytoplasm of the cell using methods known to one with skill in the art. The NLS-PNA-NA translocates from the cytoplasm into the nucleus through the action of the NLS. In other embodiments, an MTS-NLS-PNA is annealed to a region of the nucleic acid and transferred into the nuclear compartment of a cell simply by putting the MTS-NLS-PNA-NA in contact with the cell.
Accordingly, the present invention can include a nucleic acid. The nucleic acid can be single-stranded, double-stranded, DNA, RNA, an expression vector, an expression vector with an insert, a plasmid, a circular nucleic acid, a linear nucleic acid, a viral vector, a therapeutic vector, and the like. In certain embodiments, the nucleic acid comprises an antisense nucleic acid targeted to complementary sequences in the nucleus. Specifically, the bridging of introns, exons, and intron-exon boundaries is contemplated with antisense strands or antisense encoding vectors. In certain embodiments, the nucleic acid encodes an expression product, wherein the expression product comprises a peptide, a polypeptide, a protein, a fusion protein, or an antisense nucleic acid. In certain embodiments, an NLS-PNA (or an MTS-NLS-PNA as described herein) has an antisense activity, wherein the antisense activity is localized in the nucleus, not in the cytoplasm because the NLS directs the activity to the nucleus and retains it in the nucleus. U.S. Pat. No. 5,700,922 to Cook, incorporated herein by reference; describes the use of PNA-DNA-PNA chimeras in non-nuclear antisense reactions. In certain embodiments wherein an antisense nucleic acid is expressed from a nucleic acid annealed to an NLS-PNA (or an MTS-NLS-PNA as described herein), the antisense reaction can take place in any compartment of the cell or even outside of the cell. In certain embodiments, the nucleic acid encodes a gene, a reporter gene, a gene fusion, a transgene, or a therapeutic gene. A particularly useful nucleic acid, comprises a double-stranded expression vector with an insert under the control of a cytomegalovirus (CMV) promoter. The preferred PNA sequence, for use with this nucleic acid, comprises the peptide nucleic acid sequence ACTGCCCA which is complementary to a region on the non-coding strand of the CMV promoter.